Quality control method and kit for detecting human KRAS gene variation based on high-throughput sequencing
A gene mutation and kit technology, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to reduce the cost of detection quality control and improve the efficiency.
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Embodiment 1
[0039] Example 1: Preparation of Quality Control Products for Human Circulating Tumor DNA KRAS Gene Variation Detection
[0040] 1.1. Seven commonly used stable human tumor cell lines HOP-62, HCT-15, AGS, NCI-H1573, COR-L23, NCI-H1355 and SW1417 were purchased from ATCC. The genomic DNA of each cell line was sequenced by the Sanger method, and the heterozygous and homozygous mutation sites confirmed by the Sanger method were used as positive control sites. It has been verified that there are 6 positive mutation sites in total.
[0041] 1.2. The seven human tumor cell lines were cultured in a special medium, culture conditions: 37°C constant temperature, 5% CO 2 , humidity 50%. Cultivate until the cell density reaches 80-90% of the area of the culture dish, collect the cells in the logarithmic phase of growth, centrifuge at 1500g at 4°C for 5min, take the precipitate and discard the supernatant.
[0042] 1.3. Resuspend in pre-cooled PBS solution, centrifuge at 5000rpm at 4...
Embodiment 2
[0046] Example 2: Preparation of Human Circulating Tumor DNA KRAS Gene Variation Detection Kit
[0047] 2.1. Design and synthesize multiple capture probes for different target regions on the human circulating tumor DNA KRAS gene. The collection of all capture probes can cover all coding exon regions and exon-intron junction regions of the human KRAS gene; The capture probe is labeled with biotin; the sequence of the capture probe is shown in SEQ ID NO: 1-37 in the sequence listing.
[0048] 2.2. Mix all the capture probes shown in SEQ ID NO: 1-37 in the sequence listing together in the same ratio, and dilute the mixture to a working concentration of 1.5 PM (PM = picomole / liter), and store at -20°C.
[0049] 2.3. Separately pack the capture probe mixture and the quality control obtained in Example 1.
[0050] 2.4. Prepare instructions, outer packaging, assemble and seal.
[0051] 2.5. The amount of capture probe mixture used is 6 μL / 3 reactions, 12 μL / 6 reactions, 24 μL / 12 re...
Embodiment 3
[0052] Example 3: Sequencing detection of human circulating tumor DNA KRAS gene variation
[0053] The instrument used for sequencing in this example is NextSeq CN500, an automatic gene sequence analyzer, and high-throughput sequencing was performed on the Illumina platform.
[0054] The preparation method of the quality control product is the same as that in Example 1.
[0055] 3.1. Human cfDNA was extracted from 10 positive plasma samples, and quality control products were used directly without extraction.
[0056] 3.2. Library construction
[0057] 3.2.1. End Repair
[0058] Add 30ng of extracted cfDNA sample or quality control to 0.2ml PCR reaction tube, make up to 50μL with Low EDTA TE, vortex to mix, and centrifuge briefly. Add 1 μL of end-repair enzyme mixture and 6 μL of end-repair reaction buffer, vortex and mix briefly, and then incubate at 37°C for 5 minutes, then incubate at 65°C for 30 minutes, and then at 37°C for 5 minutes for end-repair. After the reaction,...
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